Background <p>Traumatic optic neuropathy leads to irreversible vision loss due to ischemia and inflammation-induced apoptosis of optic-nerve axons. Models of retinal ganglion cell and optic nerve degeneration involve induction by chemicals (cobalt chloride, LPS, and N-methyl-D-aspartate), mechanical stress (optic nerve compression, light), and ischemia (transient retinal ischemia). Although exposure of R28 cells, retinal progenitor cells (RPCs), to severe hypoxia can cause cell damage and alter mitochondrial metabolism, the effects and mechanisms of hypoxia on these cells are unclear.</p> Methods <p>In this study, CoCl₂ was used to cause hypoxic damage to R28 cells, which were subsequently transferred to a hypoxic chamber. The levels of proteins related to hypoxia, mitochondrial homeostasis, neuro-regeneration, and retinal ganglion-like cell markers were investigated by immunoblotting. Additionally, the expression levels of genes associated with apoptosis and ferroptosis were evaluated. Mitochondrial respiration, glycolysis, and ATP synthesis were analyzed in the CoCl₂-treated group and CoCl₂-treated with 0.3% hypoxic chamber group using the Seahorse XF.</p> Results <p>As a result of transferring to a 0.3% hypoxia chamber after CoCl₂-induced hypoxia damage, the viability of RPCs increased compared to that of the control group. In addition, mitochondrial ATP production, which was reduced by CoCl₂, was recovered in a 0.3% hypoxia chamber, and expression levels of ferroptosis-associated genes were increased. In addition, decreased levels of the nerve regeneration-related markers were recovered.</p> Conclusions <p>Thus, hypoxic conditions are involved in inducing and recovering cell death or ferroptosis through mitochondrial regulation in RPCs. Therefore, we suggest that a 0.3% O₂ hypoxic chamber has significant neuroprotective potential.</p>